Identification by hydrogen/deuterium exchange of structural changes in tyrosine hydroxylase associated with regulation

Shanzhi Wang, Giri R. Sura, Lawrence J. Dangott, Paul F. Fitzpatrick

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by 3 orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrate access; this conformation is favored by phosphorylation of Ser40.

Original languageEnglish (US)
Pages (from-to)4972-4979
Number of pages8
JournalBiochemistry
Volume48
Issue number22
DOIs
StatePublished - Jun 9 2009
Externally publishedYes

Fingerprint

Phosphorylation
Deuterium
Tyrosine 3-Monooxygenase
Conformations
Hydrogen
Dopamine
Catalytic Domain
Catecholamines
Peptides
Enzymes
Serine
Mass spectrometry
Digestion
Mass Spectrometry
Kinetics
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification by hydrogen/deuterium exchange of structural changes in tyrosine hydroxylase associated with regulation. / Wang, Shanzhi; Sura, Giri R.; Dangott, Lawrence J.; Fitzpatrick, Paul F.

In: Biochemistry, Vol. 48, No. 22, 09.06.2009, p. 4972-4979.

Research output: Contribution to journalArticle

Wang, Shanzhi ; Sura, Giri R. ; Dangott, Lawrence J. ; Fitzpatrick, Paul F. / Identification by hydrogen/deuterium exchange of structural changes in tyrosine hydroxylase associated with regulation. In: Biochemistry. 2009 ; Vol. 48, No. 22. pp. 4972-4979.
@article{fdf6ea77853b4ca5b394bb1c7203b09d,
title = "Identification by hydrogen/deuterium exchange of structural changes in tyrosine hydroxylase associated with regulation",
abstract = "The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by 3 orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrate access; this conformation is favored by phosphorylation of Ser40.",
author = "Shanzhi Wang and Sura, {Giri R.} and Dangott, {Lawrence J.} and Fitzpatrick, {Paul F.}",
year = "2009",
month = "6",
day = "9",
doi = "10.1021/bi9004254",
language = "English (US)",
volume = "48",
pages = "4972--4979",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "22",

}

TY - JOUR

T1 - Identification by hydrogen/deuterium exchange of structural changes in tyrosine hydroxylase associated with regulation

AU - Wang, Shanzhi

AU - Sura, Giri R.

AU - Dangott, Lawrence J.

AU - Fitzpatrick, Paul F.

PY - 2009/6/9

Y1 - 2009/6/9

N2 - The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by 3 orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrate access; this conformation is favored by phosphorylation of Ser40.

AB - The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by 3 orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrate access; this conformation is favored by phosphorylation of Ser40.

UR - http://www.scopus.com/inward/record.url?scp=66649111062&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=66649111062&partnerID=8YFLogxK

U2 - 10.1021/bi9004254

DO - 10.1021/bi9004254

M3 - Article

VL - 48

SP - 4972

EP - 4979

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 22

ER -